Objective:

This is the second use of the ZymoBiomics DNA/RNA Prep minikit. After the first experimental concentrations and 260/280 values were less than optimal, the aim is to test the kit using bulk samples directly frozen on dry ice

Broader Context

This extraction and the previous entry were done on sediments collected from Bodega in July of 2021. We hope to be able to optimize this protocols in order to use on these samples containing high volumes of humic substances.

Protocol

1. Samples were defrosted on ice and vortexed to mix well.
2. 200ul (0.2g) of sediment were added to the zymobiomics lysing matrix tubes with 500ul of DNA/RNA shield
3. samples were bead beat at 6.5 m/s for 1 minute with a 5 minute rest for 5 cycles
4. samples were centrifuged at 10,000 rpm for 30 seconds at room temperature
5. 300ul of supernatant was transfered to a nuclease free tube where 600ul of lysis buffer was added and pipetted up and down to mix.
6. the total volume was transferred to the yellow spin column and centrifuged at 10,000rpm for 30 seconds.
7. the spin column was placed in a new collection tube and set aside
8. 900ul of 100% EtOH was added to the eluate and pipetted up and down to mix
9. 1/2 this mixture was tranferred to the green spin column and centrifuged at 10,000rpm for 30 seconds; the eluate is discarded
10. step 9 was repeated a second time to transfer the other half of the ETOH eluate mixture

11. 80ul of DNAse Mix ( 75ul DNAse prep buffer and 5 ul of DNAse I/ sample) were added to the spin column and set aside to incubate for 15 minutes

12. while the RNA sample was incubating the DNA in the yellow spin column previously set aside was processed
13. 400ul of DNA/RNA prep Buffer was added to the column and centrifuged at 10,000 rpm for 30 seconds
14. 700ul of DNA/RNA prep Buffer was added to the column and centrifuged at 10,000 rpm for 30 seconds
15. 400ul of DNA/RNA wash Buffer was added to the column and centrifuged at 10,000 rpm for 30 seconds

16. spin column was transfered to a clean 1.5ml microcentrifuge tube and 50ul of of Nuclease free water was added to the column and incubated for 5 minutes before centrifuging at 10,000 rpm for 30 seconds for two rounds ( final elution volume 100 ul)

17. once the RNA on the green column finished its incubation period, steps 13-16 were repeated on the green column to process the RNA.
18. HRC filters (white column) were prepped by adding 600 ul of HRC Prep solutions and centrifuging at 8,000 rpm for 3 minutes
19. All eluted samples were added onto HRC filters in new 1.5ml tubes and centrifuged at 16,000 rpm for 3 minutes.
20. samples were frozen at -80C for later down stream processing.

Raw Data

Conclusions

Clearly based on this data the extractions were not very successful, it seems like if anything having samples already frozen in zymo will be more effective. However, the bulk sediment being used in these experiments has come from a single falcon tube and been unfrozen and refrozen several times and so any NA present on collection may have been degraded. Best next step is to rerun with the samples in both the kit and through chemical extraction from non-bulk sediment that has yet to be defrosted to confirm if degradation may be causing part of the issue.